Summary

Scar, a member of the WASp protein family, was discovered in
Dictyostelium discoideum during a genetic screen for second-site
mutations that suppressed a developmental defect. Disruption of the
scar gene reduced the levels of cellular F-actin by 50%. To
investigate the role of Scar in endocytosis, phagocytosis and endocytic
membrane trafficking, processes that depend on actin polymerization, we have
analyzed a Dictyostelium cell line that is genetically null for Scar.
Rates of fluid phase macropinocytosis and phagocytosis are significantly
reduced in the scar- cell-line. In addition, exocytosis of
fluid phase is delayed in these cells and movement of fluid phase from
lysosomes to post-lysosomes is also delayed. Inhibition of actin
polymerization with cytochalasin A resulted in similar phenotypes, suggesting
that Scar-mediated polymerization of the actin cytoskeleton was important in
the regulation of these processes. Supporting this conclusion, fluorescence
microscopy revealed that some endo-lysosomes were ringed with F-actin in
control cells but no F-actin was detected associated with endo-lysosomes in
Scar null cells. Disruption of the two genes encoding the actin monomer
sequestering protein profilin in wild-type cells causes defects in the rate of
pinocytosis and fluid phase efflux. Consistent with a predicted physical
interaction between Scar and profilin, disrupting the scar gene in
the profilin null background results in greater decreases in the rate of fluid
phase internalization and fluid phase release compared to either mutant alone.
Taken together, these data support a model in which Scar and profilin
functionally interact to regulate internalization of fluid and particles and
later steps in the endosomal pathway, probably through regulation of actin
cytoskeleton polymerization.

INTRODUCTION

Wiskott-Aldrich syndrome is an X-linked immunodeficiency disease that
results from the failure of both T and B cell function and additional defects
in monocyte chemotaxis (Ochs et al.,
1980). As a result, patients
with this disease not only have compromised immune function, but also have
increased incidence of thrombocytopenia and are more susceptible to lymphomas
and leukemias (Kirchhausen and Rosen,
1996). Recently, the gene
responsible for this disease was identified (Derry et al.,
1994) and found to encode a 54
kDa protein. The WASp protein can associate with filamentous (F)-actin and is
hypothesized to participate in Cdc42 mediated signal transduction pathways
that regulate the polymerization of F-actin (Aspenstrom et al.,
1996; Kolluri et al.,
1996; Symons et al.,
1996). Although the originally
identified WASp protein was shown to be expressed only in cells of
hematopoietic origin (Derry et al.,
1994), another closely related
protein, N-WASP, was later found to be ubiquitously expressed but enriched in
neural tissue (Miki et al.,
1996).

It has been proposed that the various forms of WASp interact with several
other proteins in order to induce the polymerization of F-actin. For instance,
it was shown that, through its proline-rich domains, WASp can bind to the
adaptor proteins Nck and Grb2 (Rivero-Leczano et al.,
1995; She et al.,
1997). In addition, WASp can
bind a novel protein termed WIP (Wiskott-Aldrich syndrome protein-interacting
protein) (Ramesh et al.,
1997). Verprolin, a WIP
ortholog, in turn, was demonstrated to control cell polarity in yeast (Vaduva
et al., 1999). WIP also binds
to the adaptor protein Nck, suggesting that Nck, WASp and Wip might form
complexes that regulate the actin polymerization machinery (Anton et al.,
1998). Further evidence for the
involvement of Wip/WASp in actin polymerization has come from experiments
showing that site-directed mutations in WASp impair its interaction with Wip
and might lead to Wiskott-Aldrich syndrome (Stewart et al.,
1999). Finally, it has also
been established that N-WASp can bind actin monomers and sequester the
profilin through its polyproline domain, and that this association leads to
plasma membrane alterations (Suetsugu et al.,
1998).

In a genetic screen for second-site suppressors of a mutation in one of the
cAMP receptors, another protein that has significant homology to WASp, Scar,
was identified in D. discoideum (Bear et al.,
1998). Subsequently, a Scar
homolog, termed h-Scar1 or Wave, was identified in humans and other
vertebrates (Bear et al., 1998;
Miki et al., 1998). When
scar was disrupted in Dictyostelium, it was able to restore
the normal development to cells that were null for the cAMP receptor cAR2
(Saxe et al., 1993). When
scar was disrupted in a wild-type background, both morphogenetic and
actin cytoskeletal defects were seen in Dictyostelium (Bear et al.,
1998; C.L.S. et al.,
unpublished), indicating that Scar might behave in a similar manner to the
closely related protein WASp. Interestingly, alignment of the two proteins
showed that Scar shares many of the same domains as WASp, including a
C-terminal acidic domain, a short WASp-homology domain (shown to interact with
actin) and a polyproline rich domain (shown to interact with profilin).
However, unlike WASp, Scar family members do not contain a Cdc42/Rac1
interactive binding (CRIB) domain and, instead have a novel protein region,
termed a Scar homology domain (SHD). Finally, whereas WASp contains an
N-terminal domain related to pleckstrin homology domains, Scar does not. This
evidence suggests that WASp and Scar have both overlapping and distinct
functions (reviewed in Mullins,
2000).

Recent data (Machesky et al.,
1999; Rohatgi et al.,
1999) have provided perhaps
the most compelling evidence for a role for Scar/WASp in regulating actin
cytoskeleton organization. These studies confirmed that Scar/WASp bound to and
stimulated the activity of the Arp2/3 complex, a seven protein complex that
was previously shown to be important in the dendritic nucleation involved in
forming branching F-actin (Mullins et al.,
1997; Mullins et al.,
1998; Welch et al.,
1997). The proposed dendritic
nucleation model suggests that Arp2/3 associates with actin on the side of
existing filaments and acts as a cap for the pointed end of a newly formed
actin filament. Scar is believed to bind to the complex and activate the
nucleation activity of Arp2/3, causing the nascent filament to grow in the
barbed-end direction. Thus, cells can control the localized polymerization of
actin to form lamellipodia by adding on to already-existing filaments (Weiner
et al., 1999; Svitkina and
Borisy, 1999).

The Arp2/3 complex was originally discovered by searching for binding
partners for the actin binding protein profilin in Acanthamoeba
(Machesky et al., 1994). It
was subsequently found that one of the subunits of the Arp2/3 complex could
bind to actin (Mullins et al.,
1997). Around the same time,
it was found (Suetsugu et al.,
1998) that N-WASp could also
bind profilin at its polyproline stretch of amino acids, suggesting the
possibility that Scar or WASp binds to profilin-actin to recruit it to the
Arp2/3 complex. This model is probably an oversimplification, as profilin
inhibits the polymerization of F-actin in vitro in the presence of Arp2/3 and
Scar (Machesky et al.,
1999).

The exact function of profilin remains unknown, as seemingly conflicting
experimental results have been published. It is now hypothesized that at low
concentrations profilin stimulates actin assembly (Vinson et al.,
1998), while at high
concentrations it behaves as an actin monomer sequestering protein and
prevents F-actin assembly (Carlsson et al.,
1977). Deletion of the two
genes encoding profilin in Dictyostelium results in a number of
phenotypic changes (Haugwitz et al.,
1994). Motility is reduced and
mutant cells are large and contain a broad rim of cortical actin, suggesting
that profilin acts primarily to sequester monomeric actin in
Dictyostelium. In addition, the profilin null mutants show defects in
endosomal trafficking and internalization (Temesvari et al.,
2000).

Two recent papers demonstrate that WASp also plays a role in the regulation
of phagocytosis (May et al.,
2000; Lorenzi et al.,
2000). In order to gain more
insight into how WASp-like proteins might function in the regulation of
endocytosis and endo-lysosomal membrane trafficking, processes dependent on
actin regulation, we have analyzed a D. discoideum cell line in which
the gene encoding Scar has been disrupted (Bear et al.,
1998). Previous studies of
Dictyostelium have demonstrated the importance of the actin
cytoskeleton in the regulation of vesicle trafficking and endocytic pathways
(Buczynski et al., 1997;
Seastone et al., 1998; Maniak
et al., 1995; Hacker et al.,
1997; Temesvari et al.,
2000). The current studies
demonstrate that a knockout of the gene encoding Scar results in defects in
macropinocytosis and phagocytosis. In addition, the scar disruption
causes a block in the trafficking and release of fluid phase from cells.
Finally, disrupting the scar gene in a Dictyostelium mutant
null for both profilin proteins further reduced the levels of fluid phase
pinocytosis and exocytosis observed in the profilin mutant. Taken together,
these results suggest a model in which profilin and Scar act in parallel
and/or in concert to regulate the polymerization of F-actin that is critical
for multiple endocytic processes.

Profilin/Scar triple null cells
(pI-/II-/scar-) were constructed by
introducing the 9A/O7 plasmid (Bear et al.,
1998) into the profilin double
null mutant (provided by M. Schleicher) and selecting for
blasticidin-resistant transformants. The genotype was confirmed by PCR-based
detection of the Scar-blastocidin cassette as previously described (Bear et
al., 1998). These cells were
maintained on HL5 medium in tissue culture dishes or on lawns of
Klebsiella aerogenes.

Phagocytosis, fluid phase pinocytosis and exocytosis assays

For phagocytosis, fluid phase pinocytosis and exocytosis assays,
exponentially growing cells were harvested from T-175 tissue culture flasks
(Sarstedt) and resuspended in growth medium at a titer of
3×106 cells ml-1. For phagocytosis assays, cells
were exposed to 1 μm fluorescent crimson latex beads (Molecular Probes) in
HL5 medium at a concentration of 50 beads per cell. Cells and beads were
shaken at 150 rpm in 25 ml Erlenmeyer flasks for 90 minutes. At various times,
1 ml aliquots of cells were harvested by centrifugation (1000
g for 5 minutes), washed twice with cold HL5 growth medium,
and once with sucrose buffer (5 mM glycine, 100 mM sucrose, pH 8.5). Cells
were lysed with 0.5% Triton X-100 and the intracellular fluorescence was
measured by spectrofluorimetry using 625 nm wavelength for excitation and 645
nm for emission. The fluorescence of each of the samples was normalized to
total cell protein to account for any differences in cell sizes between the
strains. For fluid phase pinocytosis assays, FITC-dextran (relative molecular
mass (Mr) 70,000, Sigma) was incubated with shaking
cultures of cells in growth medium to a final concentration of 2 mg
ml-1 for 2 hours. At various times, cells were harvested and lysed
with 0.5% Triton X-100, and the intracellular fluorescence was calculated with
a spectrofluorimeter using 492 nm for excitation and 525 nm for emission. For
exocytosis assays, cells were loaded for 3 hours with 70,000
Mr FITC-dextran. The cells were washed twice with cold HL5
medium and resuspended in growth medium. At various times, the cells were
harvested, lysed with 0.5% Triton X-100, and the intracellular fluorescence
was determined as described above. To determine the percent of fluorescence
remaining in the cells, the fluorescence value at each time point was compared
with the fluorescence at time T=0, which was given a value of
100%.

pH flux assays

For pH flux assays, cells were harvested from T-175 flasks and resuspended
in HL5 growth medium containing FITC-dextran (5 mg ml-1) at a titer
of 3×106 cells ml-1. After a 10 minute pulse with
the FITC-dextran, cells were harvested by centrifugation (1000
g for 5 minutes) and resuspended in growth medium without
FITC-dextran. At various times during the chase period, 1 ml aliquots of cells
were harvested, washed twice with HL5 medium, once with MES buffer (50 mM),
and then resuspended in 1 ml MES. The ratio of the emission value at 525 nm
(after excitation at 450 nm) to that after excitation at 495 nm was calculated
and compared to a FITC fluorescence/pH standard curve to obtain the vesicular
pH.

Fluorescence microscopy

Cells were harvested from T-25 tissue culture flasks and incubated in HL5
medium with the fluid phase markers RITC-dextran (2 mg ml-1) or
FITC-dextran (2 mg ml-1). For macropinocytosis measurements, cells
were pulsed for 5 minutes while attached to coverslips. For experiments
designed to examine the steady state appearance of endolysosomal vesicles,
cells shaking in suspension were pulsed for 1 hour with FITC-dextran (2 mg
ml-1) or RITC-dextran (2 mg ml-1) and washed with HL5
growth medium. The cells were resuspended in fresh growth medium and
Lysosensor Green (Molecular Probes, Eugene, OR) was added at a dilution of
1:1000. Cells were immediately spotted onto plastic coverslips and, after 10
minutes, they were gently rinsed with HL5 medium and examined using
fluorescence microscopy.

F-actin staining of wild-type, profilin and profilin/Scar null mutants was
performed as described (Bear et al.,
1998). Briefly, growing cells
were collected, allowed to adhere to glass coverslips, washed with PBS and
stained with PBS containing 400 nM TRITC-phalloidin (Sigma). Cells were viewed
on a Model 510 confocal microscope (Carl Zeiss) or an Olympus Ax70
epifluorescence microscope. F-actin was visualized in vivo using cell lines
expressing GFP-ABD, a fusion protein previously shown to bind intracellular
F-actin with high specificity.

Lysosomal hydrolase secretion assay

Cells were cultured in HL5 growth medium and were harvested from T-175
flasks, washed and the steady state intracellular and extracellular levels of α
-mannosidase activity were assayed as described (Seastone et al.,
1998).

RESULTS

Disruption of scar reduces the rates of phagocytosis and
fluid phase endocytosis

To determine the role of Scar in regulating phagocytosis and fluid phase
endocytosis, processes previously demonstrated to require actin polymerization
(Lamaze et al, 1997; Maniak et
al., 1995; Hacker et al.,
1997), wild-type and Scar null
cells were incubated with 1 μm fluorescent latex beads or FITC-dextran. At
various times, cells were washed and intracellular fluorescence was measured.
As shown in Fig. 1A, the rate
of phagocytosis of the latex particles was decreased by 80% in
scar- cells compared to control Ax3 cells and the rate of
uptake of the fluid phase marker FITC-dextran was decreased by 40%
(Fig. 1B). The rate of uptake
of fluorescently labeled Escherichia coli was also reduced by 80% in
the Scar null strain as compared to control cells (results not shown),
indicating that the phagocytic defect is not specific for latex particles.

scar- cells are defective in phagocytosis, fluid phase
pinocytosis and macropinocytosis. To determine the rates of phagocytosis,
cells were incubated with 1 μm fluorescent latex beads or FITC-dextran for
the indicated times and the intracellular fluorescence was calculated using a
spectrofluorimeter. The fluorescence value at each time point was normalized
to protein load to account for any difference in cell size among the strains.
(A) scar- cells internalized beads at a rate two to three
times less than that of control cells, indicating that the mutant was
defective in phagocytosis (n=6). (B) scar- cells
internalized FITC-dextran at half the rate of control cells, indicating that
the mutant also defective in fluid phase pinocytosis (n=5). (C-F)
Cells were incubated with 2 mg ml-1 FITC-dextran for 10 minutes,
washed twice in fresh HL5 growth medium, spotted onto plastic coverslips and
examined using phase contrast (C,E) or fluorescence (D,F) microscopy. Control
cells (C,D) contained many large macropinosomal vesicles (arrows), whereas the
scar- cells (E,F) contained no large macropinosomes. Bar,
2.5 μm.

Much of the uptake of fluid phase that occurs in Dictyostelium is
through the actin-based process of macropinocytosis (Hacker et al.,
1997). In order to determine
whether the decrease in fluid phase uptake was due to a decrease in
macropinocytosis, we incubated cells attached to coverslips with the fluid
phase marker FITC-dextran for 5 minutes, fixed cells with 1% formaldehyde and
examined them by fluorescence microscopy. As shown in
Fig. 1, in most of the control
Ax3 cells after a 5 minute pulse, fluid phase resided in one or two
macropinosomes that range in size between 2 μm and 3 μm in diameter
(Fig. 1C,D). However, in
scar- cells, large fluid phase filled vesicles were
essentially absent and only a few, much smaller, vesicles were observed
(Fig. 1E,F). Therefore, it
appeared that the scar- cells were defective in
macropinocytosis as well as in phagocytosis.

Scar null cells are defective in late endo-lysosomal trafficking
events

Within minutes of internalization, fluid is transported to acidic lysosomes
(Aubry et al., 1993), followed
by transported from lysosomes to neutral pH post-lysosomes (Padh et al.,
1993), from where it is
released into the extracellular milieu beginning approximately 45 minutes
after internalization. To determine whether Scar played a role in regulating
the release of fluid phase material from the endo-lysosomal system of
Dictyostelium, wild-type and mutant cells were allowed to internalize
FITC dextran for 3 hours (to load all endocytic compartments completely),
washed and placed back into marker-free growth medium to initiate the chase.
At various time points, cells were harvested and washed, and the fluorescence
remaining in the cells was measured using a spectrofluorimeter.
Fig. 2A indicates that the rate
of release of fluid phase from wild-type cells was significantly higher than
that observed for the mutant cells. For instance, after 50 minutes of chase,
wild-type cells retained 50% of the internalized FITC-dextran, whereas
scar- cells still retained 75%. The
scar- cells did not release 50% of the FITC-dextran until
90 minutes of chase, at which time the control cells had already released
80%.

scar- cells are defective in fluid phase exocytosis. To
examine exocytosis, wild-type and scar- cells were loaded
with FITC-dextran for 3 hours, washed and allowed to efflux FITC-dextran for
the indicated times prior to harvesting and fluorescence measurement. The
percentage FITC-dextran remaining inside the cell was calculating by comparing
the fluorescence values at each of the time points to the value at time
T=0. (A) The average of four independent experiments, showing that
scar- cells are defective in exocytosis. Whereas wild-type
cells have released 50% of the FITC-dextran from the cell by 50 minutes,
scar- cells required nearly twice as long (90 minutes) to
exocytose 50% of the FITC-dextran. (B) The vesicular pH was calculated over
time as described in Materials and Methods. Fluid phase entered acidic
vesicles rapidly (within 10 minutes into the chase period in both control Ax3
and scar- cells). However, the fluid phase only slowly
left lysosomes in scar- cells and did not reach more
neutral pH post-lysosomes until after 60 minutes into the chase period,
whereas, in wild-type cells, the fluid phase entered the post-lysosomes within
45 minutes.

This result indicated that there was a delay in the release of fluid from
the endo-lysosomal system in scar- cells. To determine if
this delay occurred before or after fluid phase reached acidic lysosomes,
cells were pulsed with FITC-dextran for 10 minutes, washed and placed into
fresh growth medium. At various time points, the fluorescence of intracellular
FITC-dextran (525 nm) was measured after excitation at 450 and 495 nm; the
ratio of fluorescence was converted to pH using a standard curve. As shown in
Fig. 2B, fluorescence ratio
measurements indicated that fluid phase entered the most acidic compartments
(pH <5) 5-10 minutes after initiating the chase period in both control and
scar- cells. This result is in accordance with previously
published studies showing that fluid phase normally enters acidic lysosomes
within minutes after internalization (Padh et al.,
1993). However, in control
cells, the fluid phase marker rapidly entered more neutral pH compartments (pH >
5.5), whereas, in scar- cells, this process was
delayed, suggesting that fluid phase was retained in acidic compartments
(Fig. 2B).

As an alternative approach to determine whether fluid phase accumulated in
acidic lysosomes in the mutant cells, we incubated scar-
and wild-type cells with FITC-dextran for 1 hour and then allowed cells to
attach to coverslips. Subsequent examination by fluorescence microscopy
revealed that control cells (Fig.
3A,B) contained fluorescent vesicles of many different sizes
including large post-lysosomes and macropinosomes (vesicles >0.5 μm
marked with arrows) and smaller lysosomes (<0.5 μm, marked with an arrow
head). We have previously demonstrated that these large, more fluorescent
vesicles are much less acidic than the smaller less fluorescent lysosomes
(Buczynski et al., 1997). By
contrast, scar- cells contained primarily vesicles the
size of lysosomes (Fig. 3C,D).
To determine whether these smaller vesicles in the scar-
cells were acidic and thus further support the idea that they were lysosomes,
we incubated mutant cells attached to coverslips with RITC-dextran for 1 hour
and then added Lysosensor Green (Molecular Probes), a chemical that only
fluoresces in acidic compartments. As expected, most of the vesicles in the
scar- cells that were labeled with RITC-dextran also
labeled positively with Lysosensor Green (compare
Fig. 3E,F), indicating that
there were few near-neutral endo-lysosomal vesicles in these cells. By
contrast, as reported previously (Buczynski et al.,
1997), the large RITC-dextran
positive vesicles in the control strain did not label with Lysosensor Green,
which is consistent with the presence of macropinosomes and post-lysosome
organelles (data not shown). Taken together, these results are consistent with
the hypothesis that Scar is involved in the regulation of the transport of
fluid from acidic lysosomes to post-lysosomes.

scar- cells contain mostly small acidic vesicles. Phase
contrast (A,C) and fluorescent (B,D) microscopic images of control (A,B) and
scar- cells (C,D). (A-D) Cells were incubated with
FITC-dextran for 1 hour, washed and spotted onto coverslips prior to
examination. In control cells (A,B), vesicles of many different sizes were
present, including large post-lysosomal and macropinosomal vesicles (arrows).
By contrast, scar- cells contained primarily smaller
vesicles. (E,F) scar- cells were incubated with
RITC-dextran for 1 hour, washed and further incubated with Lysosensor Green
for 10 minutes. After spotting cells on coverslips, they were examined using
the red channel (E) or the green channel (F) of the fluorescence microscope.
Most of the RITC-dextran-positive vesicles (E) also stained with Lysosensor
Green (F), indicating that most of the vesicles in the
scar- cells were acidic. Bar, 5 μm.

F-actin associates with endo-lysosomes in wild-type cells but not
Scar null cells

Polymerization of the actin cytoskeleton plays an important role in the
regulation of the endo-lysosomal system of several different cell types
including mammalian cells, yeast and Dictyostelium (Lamaze et al.,
1997; Kubler and Riezman,
1993; Jenne et al.,
1998; Maniak et al.,
1995; Hacker et al.,
1997; Seastone et al.,
1998; Seastone et al.,
1999). Scar might act to
regulate the endo-lysosomal system by controlling the polymerization of actin
through recruitment and/or activation of the Arp2/3 complex (Machesky et al.,
1999). Scar could bind to
endo-lysosomes and initiate the polymerization of F-actin, which is necessary
for endosomal fluid transport. In fact, a previous publication demonstrated
that N-WASp associated with endo-lysosomes and stimulated F-actin
polymerization that propelled lysosomes (Taunton et al.,
2000). A prediction of this
hypothesis is that Scar null cells should contain significantly fewer
endo-lysosomes ringed with F-actin than control cells. To test this
hypothesis, we first analyzed control cells expressing GFP-ABD protein, which
has previously been demonstrated to bind to F-actin (Pang et al.,
1998). Cells were allowed to
internalize Texas Red (TR)-dextran for 1 hour to load the endosomal
compartment. Fig. 4 shows that
control cells contain a few TR-dextran-positive endo-lysosomes
(Fig. 4B) ringed with GFP-ABD
(Fig. 4A). Although, in most
cells, only a small proportion of the endo-lysosomes were ringed with GFP-ABD,
all of the GFP-ABD-positive vesicles contained TR-dextran, supporting the
concept that GFP-ABD positive vesicles were endo-lysosomal.

F-Actin rings endo-lysosomes in control but not in
scar- cells. Cells expressing GFP-ABD (A) were incubated
with RITC-dextran in growth medium for 1 hour (B), recovered by centrifugation
and fixed with formaldehyde prior to visualization using a fluorescence
microscope. These two panels show that all the vesicles ringed with F-actin
were endo-lysosomal in nature. (C-F) Control cells (C,D) and
scar- cells (E,F) were fixed and decorated with
fluorescent phalloidin to visualize F-actin.

Next, control and Scar null cells were fixed and stained with TR-phalloidin
to visualize F-actin. Fluorescent dextran was not included in this experiment
because the fixation conditions necessary to visualize F-actin preclude
retention of fluid in vesicles. Control cells contained on average between one
and three F-actin-positive vesicular structures
(Fig. 4C,D), demonstrated above
to be endosomes. By contrast, no F-actin ringed structures were observed in >
100 Scar null cells (Fig.
4E,F), suggesting that Scar is necessary for the recruitment or
polymerization of F-actin to endo-lysosomes.

Three approaches were used to determine whether Scar associated with
endo-lysosomes. In the first approach, cells expressing GFP-Scar were loaded
with TR-dextran and viewed using a fluorescence microscope. The GFP-Scar
distribution appeared cytosolic, comparable to GFP expressed alone (results
not shown). This distribution is not surprising because >90% of Scar is
cytosolic (based on subcellular fractionation). No TR-dextran-positive
vesicles were ringed with GFP-Scar, although the low level of expression of
GFP-Scar might have prevented detection. Unfortunately, higher levels of
expression of GFP-Scar proved toxic to cells. In the second approach,
immunofluorescence microscopic approaches were used define the location of
Scar in cells and, as observed for GFP-Scar, the fluorescence appeared diffuse
and cytosolic in nature. In the third approach, cells loaded with iron-dextran
were fractionated on a magnetic column using a published technique (Temesvari
et al., 1994) that has been
demonstrated to yield highly pure endosomes and lysosomes. As demonstrated by
western blot analysis, Scar was found to associate with endo-lysosomes but the
level of association was not much greater than that observed for other
cytosolic markers (results not shown). Together, these results suggest that
Scar does not associate stably with endo-lysosomes. However, our results do
not exclude the possibility that Scar associates with vesicles in a transient
manner, and this association is not very stable. If this is true, the methods
used above to detect this interaction would not have yielded positive
results.

The above data suggest that, by regulating F-actin polymerization, Scar
plays major role in internalization of fluid and particles, and trafficking
along the endosomal pathway. This hypothesis predicts that inhibition of actin
polymerization by chemical means might result in phenotypic changes comparable
to those observed in scar- cells. Accordingly, wild-type
cells were treated with the drug cytochalasin A, which prevents polymerization
of F-actin (Himes et al.,
1976). Under these conditions,
and as reported by others (Hacker et al.,
1997), we observed a
dose-dependent inhibition in the rate of fluid phase endocytosis and
phagocytosis (results not shown). Interestingly, the 50% inhibitory
concentration of cytochalasin A for endocytosis was much lower than for
phagocytosis (0.2 μM vs 2 μM), suggesting that, in
Dictyostelium, a drug that prevents polymerization from the barbed
ends of F-actin has a greater negative affect on fluid phase internalization
than on the uptake of particles. Treating cells with cytochalasin A also
caused a dose-dependent delay in exocytosis of fluid and inhibited the
movement of fluid phase into and from acidic to more neutral pH compartments,
as previously observed by others (Rauchenberger et al.,
1997). Together, these results
are consistent with the hypothesis that Scar functions to regulate both
internalization of fluid and particles, and endosomal trafficking, perhaps by
regulating the polymerization of F-actin.

Based on the available evidence indicating a potential functional
connection between Scar and profilin in the regulation of the actin
cytoskeleton (see Introduction), we constructed a triple mutant strain that
lacked both scar and the two profilin-encoding genes. The triple
mutant did not grow at all in suspension culture (data not shown), although
growth on bacterial lawns seemed to be unimpaired. Profilin mutants are
characterized by developing to the tight aggregate stage of development and
growing as large, flat cells with increased amounts of cortical F-actin
(Haugwitz et al., 1994).
scar- cells have a largely opposite phenotype, producing
multiple tipped structures during development, appearing smaller in suspension
and having reduced levels of F-actin. The triple mutant cells do not develop
beyond the tight aggregate stage (Fig.
5A,B), comparable to the developmental phenotype of the profilin
mutants. However, although vegetative cells of the triple mutant were large
(Fig. 5D) and flat, similar to
the profilin mutants (Fig. 5C),
the amount of cortical F-actin appeared to be reduced relative to the profilin
mutants, and the level appeared closer to that observed for the Scar null
(Fig. 5F). In control cells,
some of the cortical actin localizes to forming macropinocytic cups that are
absent from all the mutant cell lines. These results suggested a complicated
functional relationship between Scar and profilin, and prompted us to
characterize the triple mutant further.

Cells with the
pI-/II-/scar-
triple mutation are tight aggregate mutants and show abnormal F-actin
staining. As described in Materials and Methods, strains were produced that
were null for profilin I and profilin II and Scar.

(A,B) Development of profilin double mutants
(pI-/II-) and triple mutants
(pI-/II-/scar-),
respectively. The absence of Scar does not significantly alter the
profilin-null developmental phenotype. However, cortical F-actin staining as
visualized with TRITC-phalloidin was significantly reduced in the triple
mutant (D) relative to the double profilin mutant (C). TRITC-phalloidin
staining of wild-type (E) and Scar null cells (F) are included for comparison.
The arrow identifies a forming F-actin-rich macropinosome in control cells
(E), structures that are absent from all the mutant cell lines. Bar, 10 μ
m.

As shown in Fig. 6A, the
rate of pinocytosis was reduced by >60% in the
pI-/II- null cells, as reported
previously (Temesvari et al.,
2000), and by >80% in the
pI-/II-/scar-
mutant compared with wild-type cells (Fig.
6A). The triple mutant was also more severely defective in the
release of fluid phase (Fig.
6B) and secretion of lysosomal enzymes
(Fig. 6C) than either the
scar null (Fig. 2) or
the pI-/II- null cells alone. These
data suggest that both Scar and profilin play a positive role in fluid phase
endocytosis, fluid phase exocytosis and lysosomal enzyme secretion, and their
combined absence leads to an additive decrease in the rates of these
processes. Unfortunately, attempts to measure the rate of fluid phase movement
to and from acidic vesicular compartment were unsuccessful because the triple
mutant internalized too little FITC-dextran. However, the triple mutant and
the pI-/II- null mutant accumulated
FITC-dextran in small vesicles similar in size to the acidic vesicles observed
in the scar null strain (Fig.
7). Furthermore, in the triple mutant, these small vesicles were
acidic (they accumulated Lysosensor Green), suggesting that, as observed for
the scar null mutant, fluid accumulates in acidic lysosomes in the
triple mutant (D.J.S. and J.C., unpublished).

Cells with the
pI-/II-/scar-
triple mutation are severely defective in fluid phase pinocytosis, exocytosis
and lysosomal enzyme secretion. Cells were incubated with FITC-dextran and, at
various times, the intracellular fluorescence was calculated as described in
Materials and Methods; the averages of four independent experiments are shown
(A). Alternatively, cells were loaded with FITC-dextran for 3 hours, washed
and placed back in fresh growth medium. At various times, cells were
collected, washed and the remaining intracellular FITC-dextran was measured
(B). Finally, cells growing exponentially were collected by centrifugation and
the intracellular and extracellular levels of α-mannosidase were
measured (C). (A)
pI-/II-/scar-
cells displayed a defect in pinocytosis compared with control cells, and
pI-/II-/scar-
cells showed an additive defect compared with Scar and profilin null mutants
alone, supporting the hypothesis that these two proteins interact to regulate
fluid internalization. (B) pI-/II-
cells displayed an exocytic defect: 50% of the fluid phase remained inside the
cell after 90 minutes post-chase (compared with 45 minutes for release of one
half of the fluid phase from control cells). Cells with the
pI-/II-/scar-
triple mutation showed an additive exocytic defect: 50% of the fluid phase
remained inside the cell after 150 minutes into the chase period. After 3
hours into the chase period, none of the fluid phase remained in control
cells, whereas 20% remained in the
pI-/II- cells and 40% remained inside
the pI-/II-/scar-
cells. (C) The steady state secretion rate of α-mannosidase was
calculated by comparing the extracellular enzymatic activity with the total
enzymatic activity of the cells and supernatant from pelleted cells. At steady
state, only 20% of the α-mannosidase activity remained inside the cell,
whereas 80% of the activity resided in the supernatant, owing to secretion of
the lysosomal hydrolase. In scar- cells, 40% of the α
-mannosidase activity remained inside the cell, and for the
pI-/II- cells, 30% of the enzymatic
activity remained intracellular, indicating that there was a secretion defect
in both of these strains. The
pI-/II-/scar-
cells displayed an additive secretion defect: only 15% of the α
-mannosidase activity was found in the supernatant.

Cells with the
pI-/II-/scar-
triple mutation contain mainly small acidic endosomes. To examine the
morphology of the endo-lysosomal system of
pI-/II- and
pI-/II-/scar-
mutants, cells were incubated with FITC-dextran for 1 hour, spotted on
coverslips and prepared for phase contrast (A,C,E) or fluorescence (B,D,F)
microscopy. Control cells (A,B) contained vesicles of many different sizes,
representing pinosomes, macropinosomes, lysosomes and post-lysosomes. By
contrast, pI-/II- and
pI-/II-/scar-
cells (C-F) contained only smaller vesicles that are presumed to be lysosomes,
because they stained with an acidic fluorophore (data not shown). Bar, 5 μ
m.

DISCUSSION

In this report, we present evidence suggesting that Scar regulates multiple
steps in the endo-lysosomal system of Dictyostelium. The uptake of
latex beads and bacteria (phagocytosis), and of fluid phase (micro- and
macropinocytosis), was decreased in scar- cell-lines. In
addition, the release of internalized fluid phase to and from lysosomes and
post-lysosomes in the scar null cells was inhibited, and the movement
of fluid phase from acidic to neutral pH compartments was also delayed. Actin
plays an important role in all of these endo-lysosomal processes, as
demonstrated by treatment with cytochalasin A, which prevents polymerization
of actin. Inhibition of each endocytic process was similar to that observed in
the scar null cells. Evidence was also presented suggesting that Scar
and the actin monomer sequestering protein profilin (encoded by two genes)
functionally interact with one another. Disruption of the two profilin genes
together with the Scar gene resulted in additional defects in growth, fluid
phase endocytosis and exocytosis, and the secretion of lysosomal enzymes (this
report and Temesvari et al.,
2000). Together, our results
support the hypothesis that Scar and profilin interact to regulate F-actin
polymerization, a process that plays an important role in multiple endocytic
steps.

It has recently been reported that WASp regulates Fcγ-receptor
mediated phagocytosis in peripheral blood monocytes and that WASp is recruited
to the forming phagocytic cup (May et al.,
2000; Lorenzi et al.,
2000). Our results extend this
observation to include the Scar proteins as being important in regulating
phagocytosis. Although phagocytosis rates were greatly decreased in
scar- cells, this process was still partially active.
Additional WASp-like proteins have been identified in the
Dictyostelium databases (C.L.S., unpublished;
http://dictybase.org/dicty.html) and the activity of these proteins might
partially compensate for the loss of Scar.

We also observed a small but significant decrease in the rate of uptake of
the fluid phase marker FITC-dextran, although the formation of macropinosomes
was almost completely blocked. We therefore suggest that Scar might be equally
important in the regulation of phagocytosis and macropinocytosis, and a
Scar-independent non-macropinocytic endocytic process might partially
compensate for the decrease in macropinocytosis. There is precedent for this
in animal cells (Damke et al.,
1995) and both
clathrin-dependent and clathrin-independent processes appear to operate in
Dictyostelium (Ruscetti et al.,
1994; Hacker et al.,
1997).

The absence of Scar in Dictyostelium results in a roughly 50%
reduction in the levels of F-actin (Bear et al.,
1998), suggesting that Scar
plays a positive role in actin polymerization and that F-actin dynamics are
critical in regulating macropinocytosis and phagocytosis. In support of this,
several studies have demonstrated that F-actin binding proteins and F-actin
accumulate around the forming phagocytic and macropinocytic cups (Maniak et
al., 1995; Hacker et al.,
1997; Rupper et al.,
2001). Furthermore, the
addition to cultures of cytochalasin A (an agent that prevents F-actin
polymerization from barbed ends of growing filaments) inhibited both
phagocytosis and fluid phase endocytosis (Maniak et al.,
1995; Hacker et al.,
1997). Together with previous
published studies (Machesky et al.,
1999), these data are
consistent with the proposed role for Scar as an inducer of actin
polymerization and that it is in this role that Scar affects phagocytosis,
macropinocytosis and cell motility (C.L.S., unpublished).

We also observed severe defects in the endocytic trafficking pathways of
scar- cells: control cells released internalized fluid
phase markers and lysosomal enzymes at a significantly faster rate than
scar- cells. These data were not unexpected, given that
Scar has been proposed to regulate F-actin polymerization, and that we (this
study) and others (Rauchenberger et al.,
1997) have shown that actin
polymerization might be important for a late stage of endo-lysosomal
trafficking. We propose that actin polymerization regulates a late step in
endosomal trafficking step that might involve fusion of lysosomes. Consistent
with this latter observation, we observed a greater accumulation of small
acidic vesicles in scar- cells than in control cells. The
accumulation of acidic lysosomes and a decrease in the number of
post-lysosomes has been observed previously in cell lines that were null for
DdPIK1 and DdPIK2, two of the three known
phosphatidylinositol (PtdIns) 3-kinase genes in Dictyostelium
(Buczynski et al., 1997), and
in cell lines overexpressing the Rho-like GTPase RacC (Seastone et al.,
1998). Both of these classes
of proteins were also demonstrated in these reports to regulate the dynamics
of F-actin and there appears to be a functional interaction between Scar and
RacC (D.J.S. et al., unpublished). Maniak and co-workers have demonstrated
that F-actin-binding proteins ring macropinosomes and postlysosomes but not
acidic lysosomes (Hacker et al.,
1997; Rauchenberger et al.,
1997), a result that we have
confirmed here by examining F-actin directly. Together, these results strongly
suggest that F-actin polymerization might directly regulate the fusion of
lysosomes to form post-lysosomes, a process that is dependent on Scar.

One possible way that F-actin polymerization could regulate the fusion of
lysosomes would be to facilitate the interaction between these acidic
vesicles. Taunton and co-workers (Taunton et al.,
2000) have recently found that
N-WASp is recruited to acidic vesicles (most likely endosomes and lysosomes),
and this facilitated the assembly of actin to form actin comet tails that
propel these vesicles. Scar might play a comparable role in
Dictyostelium and the motile lysosomes might collide more frequently
to trigger fusion. Consistent with this hypothesis, Scar null cells were
devoid of vesicles that were ringed with F-actin, whereas control cells
contained endo-lysosomes the size of post-lysosomes that were ringed with
F-actin. Unfortunately, using three different methods, we were not able to
detect an association between Scar and endo-lysosomes. This might mean that
Scar never associates with endo-lysosomes, a conclusion we do not favor based
on the studies alluded to above. Instead, we favor the idea that the
association of Scar with vesicles might be transient and not stable, and the
approaches we used to detect association might thus not have been optimal.

Disruption of the two Dictyostelium profilin-encoding genes
resulted in a twofold increase in F-actin (Haugwitz et al.,
1994) and a decrease in
macropinocytosis and fluid phase efflux (this report and Temesvari et al.,
2000). Deletion of the
scar gene in the profilin null background resulted in a further
decrease in endocytosis and fluid phase release. We have proposed that
macropinocytosis requires, in addition to Scar (this report), profilin
(Temesvari et al., 2000) and
the PtdIns 3-kinases, DdPIK1 and DdPIK2 (Rupper et al.,
2001). Although the exact role
of profilin has not been defined, we propose that this protein binds PtdIns
(4,5)P2 and perhaps recruits DdPIK1 and DdPIK2 to generate
PtdIns(3,4,5)P3, and this product stimulates the process
of macropinocytosis (Rupper et al.,
2001). Scar might bind to the
plasma membrane and recruit profilin and PtdIns 3-kinases to stimulate F-actin
polymerization to drive the formation of the macropinosome, and the combined
absence of both types of proteins would have a profound negative affect on
fluid phase internalization. Alternatively, profilin might interact with
nascent macropinosomal cups and recruit Scar, which could aid in actin
polymerization.

Both profilin and Scar are required for efficient efflux of internalized
fluid phase. Not surprisingly, the combined absence of Scar and profilin
resulted in greater defects in fluid phase exocytosis. This further supports
the argument that F-actin dynamics play a critical role in a late step in the
endosomal pathway leading to release of internalized fluid, and that profilin
and Scar both play a role in this process. Our hypothesis is that profilin and
Scar interact functionally, and the absence of both proteins predictably has a
more drastic affect on efflux than the absence of either protein alone.
However, our results do not preclude the possibility that both proteins act in
parallel to regulate endocytic processes.

In summary, we have provided evidence that Scar plays an important role in
the regulation of multiple steps in the endocytic membrane trafficking,
including phagocytosis, fluid phase endocytosis (particularly
macropinocytosis) and a late step in the endosomal pathway in D.
discoideum. Scar most probably has its effects through its ability to
regulate F-actin polymerization. We also found that the
actin-monomer-sequestering protein profilin played an important role in these
endo-lysosomal processes, and our evidence suggests that profilin and Scar
both functionally contribute to regulate fluid phase endocytosis and endosomal
membrane trafficking. Future studies will be directed at identifying
additional effector proteins and defining the biochemical mechanisms that
regulate these membrane trafficking events.

Acknowledgements

The work presented here was supported by a grant to JC from the NIH
(DK39232) and a grant to KS from the NIH (GM45705).

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